The overall goal of this project is to develop oligonucleotide analogs that can effectively interact with specific RNA or DNA target sequences within the HIV genome and as a consequence of these interactions repress HIV replication. We will continue to investigate novel, nuclease resistant antisense and triplex forming 2'-0- methylribo-oligonucleoside methylphosphonates (mr-OMPs) that are targeted to critical sequences in the HIV-1 genome. The project has three Specific Aims. In Specific Aim 1, linear and hairpin antisense mr-OMPs complementary to the apical stem loop of the transactivation response element, TAR, RNA or the stem IIB of Rev response element, RRE, RNA will be prepared and their interactions with TAR and RRE RNA targets will be studied by gel electrophoretic mobility shift assays (EMSA). Chimeric anti-TAR and anti-RRE mr- OMPs that contain a tract of six deoxyribonucleotides flanked by four or five 2'-O-methylribonucleosides will be studied for their ability to form RNase H cleavable hybrids with their RNA targets. Imidazole-conjugated mr-OMPs will be prepared and their ability to catalyze hydrolysis of specific phosphodiester linkages in their RNA targets will be investigated. In Specific Aim 2, triplex forming oligonucleotides (TFOs) in which 5- methylcytosine is replaced by pseudoisocytosine will be prepared and their interactions with purine tracts found in the env- and gag-genes of HIV-1 proviral DNA will be investigated. The ability of TFOs to recognize purine tracts that contain one or more pyrimidine interruptions will be explored by synthesizing derivatives of cytosine and uracil that are designed to interact with C-G base pairs in the target DNA. To enhance efficacy, TFOs conjugated with an alkylating group will be prepared and their ability to form covalent adducts with their target DNAs will be evaluated. Specific Aim 3 will explore the biochemical and biological activities of the oligonucleotides. The ability of anti-TAR or anti-RRE mr-OMPs to inhibit Tat protein-TAR or Rev peptide- RRE interactions and to inhibit Tat- or Rev-mediated gene expression in cells will be evaluated in collaboration with Dr. Tariq Rana, University of Massachusetts Medical School. The effects of TFOs on in vitro DNA transcription will be investigated. The uptake of antisense and TFO mr-OMPs by U-937 cells (human promonocytes) in culture will be characterized and the stabilities and cellular toxicities of the oligonucleotides will be investigated. The antiviral activities of the antisense and TFO mr-OMPs will be investigated in collaboration with Dr. Jeffrey Laurence, Cornell Medical Center.